Exosomes, recently re‐named “small extracellular vesicles” or “sEV,” are emerging as an intercellular communication system. Quantification of the molecular cargo exosomes carry by on‐bead flow cytometry is needed for defining their role in information transfer and in human disease. Exosomes (sEV) isolated from cell supernatants or plasma of cancer patients by size‐exclusion chromatography were captured by biotinylated antibodies specific for antigens in the exosome cargo (e.g., tetraspanins) and placed on streptavidin‐labeled beads. Detection was performed with pretitered fluorochrome‐labeled antibodies of desired specificity. The data were acquired in a conventional cytometer, and molecules of equivalent soluble fluorochrome (MESF) beads were used to quantify the number of fluorescent molecules bound per bead. Isotype antibody controls were obligatory. The mean fluorescence intensity (MFI) value of each sample was converted into MESF units, and the separation index (SI), which quantifies separation of stained and isotype control beads, was determined. Various proteins identified by labeled antibodies were quantified on the surface of tumor cell‐derived exosomes. To identify intravesicular cargo, such as cytokines or chemokines, exosomes were lysed with 0.3% Triton‐100, and the proteins in lysates were loaded on aldehyde/sulfate latex beads for flow cytometry. Examples of quantitative surface and/or intravesicular on‐bead flow cytometry for exosomes produced by various cells or present in body fluids of cancer patients are provided. On‐bead flow cytometry standardized for use with conventional cytometers is a useful method for protein detection and quantitation in exosomes isolated from supernatants of cell lines or plasma of patients with cancer. © 2020 International Society for Advancement of Cytometry

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通过珠上流式细胞术评估外泌体蛋白

外泌体，最近被重新命名为“小细胞外囊泡”或“sEV”，正在成为一种细胞间通讯系统。需要通过珠上流式细胞术对携带的分子货物外泌体进行量化，以确定它们在信息传递和人类疾病中的作用。通过尺寸排阻色谱法从癌症患者的细胞上清液或血浆中分离出的外泌体 (sEV) 被外泌体货物（例如四跨膜蛋白）中的抗原特异性生物素化抗体捕获，并放置在链霉亲和素标记的珠子上。使用具有所需特异性的预滴定荧光染料标记的抗体进行检测。数据是在传统的细胞仪中获得的，等效可溶性荧光染料 (MESF) 珠子的分子用于量化每个珠子结合的荧光分子的数量。同种型抗体对照是强制性的。将每个样品的平均荧光强度 (MFI) 值转换为 MESF 单位，并确定量化染色和同种型对照珠的分离的分离指数 (SI)。在肿瘤细胞衍生的外泌体表面上对标记抗体鉴定的各种蛋白质进行量化。为了识别囊泡内的货物，例如细胞因子或趋化因子，外泌体用 0.3% Triton-100 裂解，裂解物中的蛋白质加载到醛/硫酸盐乳胶珠上用于流式细胞术。提供了用于由各种细胞产生或存在于癌症患者体液中的外泌体的定量表面和/或囊内珠上流式细胞仪的示例。与常规细胞仪一起使用标准化的珠上流式细胞仪是一种有用的方法，用于检测和定量从癌症患者的细胞系上清液或血浆中分离的外泌体中的蛋白质。© 2020 国际细胞术进步协会